A novel mutation in the P2Y12 receptor and a function- reducing polymorphism in protease-activated receptor 1 in a patient with chronic bleeding
A novel mutation in the P2Y12 receptor and a function- reducing polymorphism in protease-activated receptor 1 in a patient with chronic bleeding
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DOI：
10.1111/jth.12539
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发表时间：
2014-05-01
影响因子：
10.4
通讯作者：
Mundell, S. J.
中科院分区：
文献类型：
--
作者：
Patel, Y. M.;Lordkipanidze, M.;Mundell, S. J.
背景：对出血患者的研究是确定人类血小板重要蛋白的功能和调控的有力途径。我们发现了一位慢性出血性疾病患者，表达纯合子P2RY12突变，预测g蛋白偶联P2Y（12）受体中精氨酸到半胱氨酸（R122C）的替代。这种突变是在DRY基序中发现的，DRY基序是g蛋白偶联受体（gpcr）中高度保守的区域，据推测在调节受体构象状态中起关键作用。目的确定R122C替代P2Y（12）功能的功能后果。患者/方法我们对一个病例和受影响的家庭成员进行了详细的表型分析。对细胞中稳定表达的变体R122C P2Y（12）也进行了分析。结果患者及其家属P2Y（12）活性明显受损，导致adp刺激的血小板聚集减少。细胞表面R122C P2Y（12）在细胞系和血小板中的表达均降低；在细胞系中，这是由于不依赖激动剂的内化，随后受体转运到溶酶体。引人注目的是，由于编码蛋白酶激活受体1 （PAR-1）的F2R基因的内含子多态性，该家族成员也表现出凝血酶诱导的血小板活化降低，这已被证明与PAR-1受体活性降低有关。我们的研究首次证实了患者存在调节血小板活性的两种刺激性GPCR通路缺失，进一步表明出血性疾病是一种复杂的特征。
BackgroundThe study of patients with bleeding problems is a powerful approach in determining the function and regulation of important proteins in human platelets. We have identified a patient with a chronic bleeding disorder expressing a homozygous P2RY12 mutation, predicting an arginine to cysteine (R122C) substitution in the G-protein-coupled P2Y(12) receptor. This mutation is found within the DRY motif, which is a highly conserved region in G-protein-coupled receptors (GPCRs) that is speculated to play a critical role in regulating receptor conformational states.ObjectivesTo determine the functional consequences of the R122C substitution for P2Y(12) function.Patient/methodsWe performed a detailed phenotypic analysis of an index case and affected family members. An analysis of the variant R122C P2Y(12) stably expressed in cells was also performed.ResultsADP-stimulated platelet aggregation was reduced as a result of a significant impairment of P2Y(12) activity in the patient and family members. Cell surface R122C P2Y(12) expression was reduced both in cell lines and in platelets; in cell lines, this was as a consequence of agonist-independent internalization followed by subsequent receptor trafficking to lysosomes. Strikingly, members of this family also showed reduced thrombin-induced platelet activation, owing to an intronic polymorphism in the F2R gene, which encodes protease-activated receptor1 (PAR-1), that has been shown to be associated with reduced PAR-1 receptor activity.ConclusionsOur study is the first to demonstrate a patient with deficits in two stimulatory GPCR pathways that regulate platelet activity, further indicating that bleeding disorders constitute a complex trait.